In the nuclei of eukaryotic cells, transcription factors and DNA repair/modifying enzymes locate particular sequences or structural signatures among billions of DNA base pairs and in the presence of many other proteins bound to DNA. The long-term goal for the research team of this project is to understand what dictates the efficiency in th processes whereby the proteins locate their target sites on DNA in order to apply this knowledge to the development of therapeutics for human diseases and disorders. The overall objective in the current project is to elucidate DNA-scanning mechanisms for zinc-finger proteins at both the molecular and atomic levels. Affinity-based engineering of Cys2-His2-type zinc fingers that exhibit desired DNA-binding specificity has gained popularity for artificial gene control/manipulation. In fact, human gene therapy based on the zinc-finger nuclease technology is in phase 1 and 2 clinical trials. However, there are an increasing number of reports that suggest kinetic defects in artificial zinc-finger proteins despite their high affinities for the taget DNA sites. This represents a bottleneck for successful therapeutic applications of artificial zinc-finger proteins. Preliminary studies by the research team suggest that this problem could be resolved via protein engineering based on knowledge of the DNA-scanning mechanisms. Recently the research team found that the Egr-1 zinc-finger protein undergoes two conformationally distinct states termed the search and recognition modes while the protein molecule scans DNA. In the current project, the research team will conduct research to test the central hypothesis that the balance between the search and recognition modes is a major determinant of the kinetic efficiency in target DNA search by zinc-finger proteins. The following three specific aims will be pursued in this project: 1) to delineate how zinc-finger proteins scan DNA; 2) to understand how zinc-finger proteins bypass obstacles on DNA; and 3) to understand how zinc-finger proteins displace other proteins from the target sites. For these specific aims, the research team will use biophysical and biochemical approaches along with mutagenesis to shift the equilibrium between the search and recognition modes. NMR spectroscopy will be used to investigate the dynamics of DNA scanning at an atomic level. Fluorescence and biochemical methods will be used to characterize the kinetic and thermodynamic properties of the zinc-finger proteins in the target DNA search process at a molecular level. The current project will substantially deepen our understanding of DNA scanning by proteins. This project will also enable improvement of kinetic properties of zinc-finger proteins, and thereby boost their applications to human therapeutics and other biomedical applications.